Substrate concentration effects on H2 production performance in a continuous stirred-tank reactor (CSTR) were investigated. Using molasses as the feeding, the CSTR system was operated at chemical oxygen demand (COD) of 2000-8000 mg/L to identify the optimal working substrate concentration. Increasing substrate concentration (2000~6000 mg/L) gave better biomass content and hydrogen production, signifying that the average cellular activity for H2 production may be enhanced as the substrate concentration increased. The overall maximal biogas and hydrogen production yield were 18.69 L and 6.01 L, respectively, both of them occurred at 6000 mg/L. The gas phase H2 content did not vary considerably regardless of changes in substrate concentration. This reflects that the CSTR was a relatively stable H2-producing system. The major soluble products from hydrogen fermentation were ethanol and acetic acid, accounting for 59% and 23% of total liquid fermentation products, respectively. Thus, the dominant H2 producers in the mixed culture belonged to acidogenic bacteria that underwent ethanol-type fermentation. However, the biomass content and hydrogen production yield tended to decrease as the substrate concentration increased to 8000 mg/L, suggesting that granular sludge formation and cellular activity for H2 production may be inhibited at high substrate concentration. Ethanol, acetic, butyric and propionic were the main liquid fermentation products with the percentages of 31%, 24%, 20% and 18%, which formed the mixed-type fermentation.